Effect of the water content on the retention and enantioselectivity of albendazole and fenbendazole sulfoxides using amylose-based chiral stationary phases in organic-aqueous conditions.

Four commercially available immobilized amylose-derived CSPs (Chiralpak IA-3, Chiralpak ID-3, Chiralpak IE-3 and Chiralpak IF-3) were used in the HPLC analysis of the chiral sulfoxides albendazole (ABZ-SO) and fenbendazole (FBZ-SO) and their in vivo sulfide precursor (ABZ and FBZ) and sulfone metabolite (ABZ-SO2 and FBZ-SO2) under organic-aqueous mode. U-shape retention maps, established by varying the water content in the acetonitrile- and ethanol-water mobile phases, were indicative of two retention mechanisms operating on the same CSP. The dual retention behavior of polysaccharide-based CSPs was exploited to design greener enantioselective and chemoselective separations in a short time frame. The enantiomers of ABZ-SO and FBZ-SO were baseline resolved with water-rich mobile phases (with the main component usually being 50-65% water in acetonitrile) on the IF-3 CSP and ethanol-water 100:5 mixture on the IA-3 and IE-3 CSPs. A simultaneous separation of ABZ (or FBZ), enantiomers of the corresponding sulfoxide and sulfone was achieved on the IA-3 using ethanol-water 100:60 (acetonitrile-water 100:100 for FBZ) as a mobile phase.

Liquid chromatography-tandem mass spectrometry method for the determination of anthelmintics in alfalfa plants.

A simple and inexpensive liquid chromatography-tandem mass spectrometric method for the determination of anthelmintics in alfalfa plants (Medicago sativa L.) was developed and validated. Anthelmintics in plant leaves and stems (green chops) were extracted with methanol/acetonitrile (7:3, v/v) followed by a concentration and clean-up step using solid-phase extraction (Strata-X, 500 mg, 6 ml cartridge). After drying with nitrogen gas, the adsorbed analytes were eluted with methanol/acetonitrile (50:50, v/v) mixture followed by 100% acetonitrile. Chromatographic separation was achieved on an Atlantis T-3 (2.1 × 100 mm × 3 µm) analytical column with a Phenomenex guard cartridge (C8, 4 × 3 mm) attached to a Waters triple quadrupole mass spectrometer operated in positive electrospray ionisation mode with selected reaction monitoring. Samples were analysed using gradient elution at a flow rate of 0.35 ml min⁻¹. The mobile phase consisted of a 10 mM ammonium formate solution in (A) water/acetonitrile (90:10, v/v) and (B) methanol/acetonitrile (50:50, v/v). The method was validated for levamisole, fenbendazole, fenbendazole sulphoxide and fenbendazole sulphone at 10, 20 and 40 µg kg⁻¹ and for eprinomectin at 20, 40 and 80 µg kg⁻¹. Limits of quantification (LOQ) were 10 µg kg⁻¹ for all analytes except eprinomectin, which had an LOQ of 20 µg kg⁻¹. The overall mean recovery in green plants was between 74.2% and 81.4% with repeatabilities ranging from 2.2% to 19.1% and reproducibilities in the range 3.8-8.7%. The validated method was applied to plant samples in a study on the behaviour of anthelmintic drugs in a soil, plant and water system.

Dexamethasone decreases plasma levels of the prochiral fenbendazole and its chiral and achiral metabolites in sheep.

1. The effect of co-administration of either short- or long-acting formulations of DXM on hepatic function and the plasma pharmacokinetic behaviour of prochiral fenbendazole (FBZ) and its metabolites was evaluated in sheep. 2. Neither DXM treatment markedly affected any of the biochemical markers of hepatic function tested. In contrast, both formulations significantly modified the plasma pharmacokinetic behaviour of FBZ and its metabolites. 3. Plasma FBZ concentrations and the associated area under the time-concentration curves were significantly lower, although the plasma detection period was longer (72 versus 48 h) in the DXM pretreated animals compared with those given FBZ alone. 4. DXM also appeared to alter the pattern of FBZ absorption, possibly through effects on abomasal pH. The shape of the plasma concentration-time curves for oxfendazole (OFZ) and fenbendazole sulphone (FBZSO(2)) were similar to FBZ, raising the possibility that DXM treatment may have altered the liver biotransformation of the parent drug. 5. The concentrations of the (+) chiral metabolite of OFZ were significantly lower in DXM pretreated animals compared with those given FBZ alone. The trend was similar for the (-) antipode, although the differences between DXM pretreated and non-pretreated animals were not statistically significant.

Effects of diet and species on the pharmacokinetics of fenbendazole in cattle.

The plasma concentration profiles of fenbendazole (FBZ), FBZ-sulphoxide (OFZ) and FBZ-sulphone were measured following intraruminal administration of FBZ at 7.5 mg/kg bodyweight in Bos taurus and B. indicus cattle offered three different diets: 100% wheaten chaff, 100% lucerne, and a 50:50 mix of these two diets. No differences between the species were apparent except for a longer time to peak plasma concentration for OFZ in the B. taurus steers fed 100% wheaten chaff. Cattle fed wheaten chaff alone gave greater areas under the concentration-time curve and longer persistence for all metabolites than when the same cattle were fed the other diets. It is concluded that the reduced rate of passage of digesta on lower-quality fibrous diets allows greater time for absorption of FBZ and its metabolites from the gut, thereby increasing systemic availability.

Effect of diet variations on the kinetic disposition of oxfendazole in sheep.

Pharmacokinetic parameters of oxfendazole (OFZ) were determined in five sheep submitted to two different feeding regimens: a poor/dry diet based on wheat straw ad libitum, and a rich/green diet based on fresh grass grazed on pasture. Animals were acclimatised to each diet before they were administered OFZ orally at a dose of 5 mg kg-1 body weight. Blood samples were taken serially between 10 min and 168 h post-administration. Plasma concentrations of OFZ and its metabolites (fenbendazole (FBZ) and fenbendazole sulphone) were quantified using an HPLC technique. Rates of absorption of OFZ and of formation of FBZ and fenbendazole sulphone were slower with the poor/dry diet; however, the area under FBZ plasma concentration vs time curve was higher when sheep were offered the low quality diet, suggesting a shift of OFZ metabolism towards FBZ formation.

The influence of a heavy infection with sensitive and resistant strains of Ostertagia circumcincta and with Trichostrongylus colubriformis on the pharmacokinetics of febantel in lambs.

Plasma concentrations of febantel and its major metabolites, fenbendazole, oxfendazole and fenbendazole sulphone, were determined after oral administration of 7.5 mg/kg febantel in lambs before and 28 days after infection with 100,000 L3 larvae of a benzimidazole (BZ)-sensitive or BZ-resistant strain of Ostertagia circumcincta or with 75,000 L3 larvae of a BZ-sensitive Trichostrongylus colubriformis strain. The febantel concentrations were always low, and in only a few samples were higher than the limit of detection. A mean decrease in the area under the curve (AUC) for the three metabolites of 10.2%, 16.4% and 4.9% in lambs infected, respectively, with BZ-sensitive O. circumcincta, BZ-resistant O. circumcincta and T. colubriformis was observed. The Cmax for all the metabolites was higher in the BZ-sensitive O. circumcincta group than in the naive sheep, while the Tmax occurred earlier. The Cmax and the Tmax values for all the metabolites were lower in the BZ-resistant O. circumcincta group than in their own naive controls. In the T. colubriformis group the Cmax values of the metabolites were lower and the Tmax occurred much later.

Plasma levels of fenbendazole metabolites in buffalo and cattle after long-term intraruminal administration.

Plasma metabolite levels were measured in buffalo and cross-bred cattle after daily intraruminal administration of fenbendazole at 0.5 mg/kg body weight. An equilibrium between the absorption of fenbendazole and disposition of its metabolites could be observed between days 3 to 6 and remained almost unchanged thereafter. The buffaloes had a lower uptake of the anthelmintic and lower plasma levels of its metabolites than in cattle. The findings were compared with those obtained after a single intraruminal therapeutic dose of fenbendazole.

Hepatic microsomal metabolism of the anthelmintic benzimidazole fenbendazole: enhanced inhibition of cytochrome P450 reactions by oxidized metabolites of the drug.

Potentiation of the anthelmintic action of benzimidazole carbamates, such as fenbendazole [methyl 5(6)-(phenylthio)-1H-benzimidazol-2-ylcarbamate], has been noted during concurrent administration of benzimidazoles that possess no intrinsic anthelmintic activity. This study investigated the possibility that inhibition of P450 enzymes by fenbendazole and its metabolites could play a role in the potentiation phenomenon. Fenbendazole underwent P450-mediated oxidation in microsomes from untreated rat liver to the sulfoxide and (4'-hydroxyphenyl)thio metabolites [2.92 and 2.87 nmol/(mg of protein.h)]. Pretreatment of rats with phenobarbital or dexamethasone enhanced sulfoxidation by 1.9- and 2.9-fold, respectively. 4'-Hydroxylation was increased slightly (by 28%) by phenobarbital and decreased slightly (by 41%) by dexamethasone. Induction also promoted further metabolism of the sulfoxide to fenbendazole sulfone. Immunoinhibition and chemical inhibition studies suggested that P450 3A proteins and the flavin-containing monooxygenase are involved in sulfoxide and sulfone formation whereas 4'-hydroxylation involved the P450s 2C11, 2C6, and 2B1, depending on the type of induction. In untreated rat liver, the sulfoxide and (4'-hydroxyphenyl)thio metabolites of fenbendazole were relatively potent inhibitors of P450-mediated androstenedione 16 alpha-, 16 beta-, and 6 beta-hydroxylation (IC50 values of 42, 36, and 74 microM, respectively); 7 alpha-hydroxylase activity was uninhibited. In contrast, fenbendazole and its sulfone metabolite were not inhibitors of these reactions. Mixed-function oxidase activities in phenobarbital-induced rat hepatic microsomes were refractory to inhibition by most compounds, but P450 1A1 mediated activities in microsomes from beta-naphthoflavone-induced rat liver were quite susceptible to inhibition by fenbendazole sulfoxide. Studies with two analogous sulfoxides yielded similar findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Qualitative and quantitative analysis of the anthelmintic fenbendazole and its metabolites in biological matrices by direct exposure probe mass spectrometry.

Methodology for the qualitative and quantitative analysis of the anthelmintic fenbendazole and its metabolites in goat feces using electron impact (EI)/direct exposure probe (DEP)/mass spectrometric (MS) and tandem mass spectrometric (MS/MS) techniques is presented. Analyses were conducted on extracts from spiked feces and feces from animals treated per os with 5 mg fenbendazole/kg, with samples being collected at zero time and at twelve hour intervals for 144 h. The results of the EI/DEP/MS quantitation of these samples are compared to those for the same samples analysed by high pressure liquid chromatography (HPLC). Mass spectral data for fenbendazole and its metabolites are presented and the advantages of the use of EI/DEP/MS and/or DEP/MS/MS over HPLC are discussed. This methodology may be used as a confirmatory method for the HPLC analysis of fenbendazole and its metabolites or may be used as a method in its own right for the rapid qualitative and quantitative analysis of these compounds.